Bypassing the restriction barrier to improve transformation in S. epidermidis

绕过限制性屏障以改善表皮葡萄球菌的转化

• 批准号: 9386188
• 负责人: Ambrose Lin Yau Cheung
• 金额: $ 20.25万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-27 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9386188
• 关键词: Adenine; Antibiotic Resistance; Bacteremia; Bacteria; Blood Circulation; Blood Vessels; Bypass; Catheters; Clinical; Cloning; Communities; Complex; Cytosine; DNA; DNA Restriction Enzymes; DNA Restriction-Modification Enzymes; Devices; Escherichia coli; Escherichia coli K12; Excision; Genes; Genetic; Genome; Genomics; Genus staphylococcus; Goals; Hip Prosthesis; Hospitals; Human; In Vitro; Individual; Indolent; Infection; Knowledge; Laboratories; Libraries; Mediating; Medical Device; Methylation; Methyltransferase; Microbial Biofilms; Mind; Modification; Morbidity - disease rate; Mucous Membrane; Nosocomial Infections; Organism; Pacemakers; Pathogenesis; Pathogenicity; Phase; Physiology; Pilot Projects; Plasmids; Positioning Attribute; Process; Prokaryotic Cells; Prosthesis; Publishing; Reporting; Research; Research Personnel; Series; Site; Skin; Specific qualifier value; Specificity; Staphylococcus aureus; Staphylococcus epidermidis; Surface; Symbiosis; System; Time; University Hospitals; Virulent; Work; antimicrobial; base; design; improved; in vivo; methylation pattern; novel strategies; pathogen; plasmid DNA; prevent; single molecule; success; uptake; virtual

Abstract Staphylococcus epidermidis (SE) ranks as one of the most common causes of nosocomial infections in US hospitals. Intravascular devices and prostheses are the usual sites of infection followed by seeding of the bloodstream. Although more indolent than S. aureus, SE infections are difficult to treat due to its persistence as biofilm on abiotic surfaces as well as its penchant for antibiotic resistance. There are two major issues in advancing our understanding of SE pathogenesis: 1) prior work has focused on two laboratory isolates, SE 1457 and RP62A while our knowledge on clinical isolates are virtually non-existent; 2) SE clinical isolates are extremely difficult to transform due to its formidable restriction barrier. As a consequence, we lack the ability to manipulate clinical isolates genetically to dissect the relevant pathogenic steps leading to colonization, biofilm formation, persistence and dissemination. With the advance of genomics and SMRT sequencing, we are in a unique position to analyze the methylation pattern of the restriction modification (RM) system based on the specificity unit (HsdS) and methylase (HsdM). There are four RM systems in SE (types I-IV). Type 1 and type IV are the major restriction barriers that prevent uptake of foreign DNA due to restriction by HsdR. We will exploit the RM system by methylating adenine in plasmid DNA with type I cognate HsdMS complex(es) in E. coli DC10B which is defective in cytosine methylation and can bypass the type IV RM system. While type IV RM system is conserved among S. aureus and SE, the type I RM system is more variable and entails at least 7 distinct groups, each with its unique target recognition motif (TRM) mediated by HsdS. These discrepancies help explain the differences in restriction among SE isolates. We hypothesize that type 1 RM system in a particular SE isolate can be completely bypassed to yield efficient transformation by cloning shuttle plasmids in DC10B that express the cognate hsdMS genes. With this goal is mind, we propose the following two specific aims: I) determine the different groups in type I RM system by performing Pacbio single molecule real time sequencing (SMRT) to detect methylated-adenine residues and the target recognition motif of the HsdMS complex in the SE genomes; II) Construction of DC10B-derived E. coli strains to enable efficient transformation into diverse SE strains. As a utility from this system, we will construct two relevant mariner transposon libraries to be available to the research community.

摘要 表皮葡萄球菌（SE）是美国医院感染最常见的原因之一 医院的血管内装置和假体是常见的感染部位，随后是植入的血管内装置。 血流虽然比S更懒惰。金黄色葡萄球菌，SE感染由于其持续性而难以治疗 作为非生物表面上的生物膜，以及它对抗生素耐药性的倾向。有两个主要问题， 推进我们对SE发病机制的了解：1）之前的工作重点是两种实验室分离株，SE 1457和RP 62 A，而我们对临床分离株的了解几乎不存在; 2）SE临床分离株 因为禁制强大，所以很难转化。因此，我们缺乏能力， 从基因上操纵临床分离株，以剖析导致定植、生物膜 形成、持续和传播。随着基因组学和SMRT测序的进步，我们正处于一个 在一个独特的位置，以分析限制性修饰（RM）系统的甲基化模式的基础上， 特异性单位（HsdS）和甲基化酶（HsdM）。在SE中有四种RM系统（类型I-IV）。类型1和类型 IV是由于HsdR的限制而阻止外源DNA摄取的主要限制屏障。我们将 通过在大肠杆菌中用I型同源HsdMS复合物甲基化质粒DNA中的腺嘌呤来开发RM系统。 coli DC 10 B中胞嘧啶甲基化缺陷，可绕过IV型RM系统。虽然第四类 RM系统在S.金黄色葡萄球菌和SE，I型RM系统更多变，至少需要 7个不同的组，每个组都有其独特的由HsdS介导的靶识别基序（TRM）。这些差异 有助于解释SE分离株之间限制性酶切的差异。我们假设，1型RM系统在一个 通过克隆穿梭质粒，可以完全绕过特定的SE分离株以产生有效的转化。 DC 10 B表达同源hsdMS基因。本着这一目标，我们提出以下两个具体建议： 目的：I）通过Pacbio单分子真实的时间测定I型RM系统中的不同基团 测序（SMRT）以检测HsdMS的甲基化腺嘌呤残基和靶识别基序 II）DC 10 B衍生的E.大肠杆菌菌株， 转化成不同的SE菌株。作为该系统的一个实用程序，我们将构建两个相关的水手 转座子库提供给研究界。